Removing phosphates - (Dec/10/2009 )

Hi, there!

I would like to know if anybody has tried to remove phosphate postranslational modifications in proteins. Till now I just knew lambda phosphatase method. Moreover, I'm trying to use lysis bufffer without phosphatase inhibitors (NaF and Vanadate, for example). I think I read sometime ago something at this respect about EDTA. Maybe somebody knows an special buffer which impairs phosphorylation... Does anybody have an idea?

Thanks very much!

-Gonzo-

EDTA is known to inhibit metalloproteases. May also chelate ions that are co-factors of certain enzymes (For example If you are gonna use CIP you should avoid EDTA in your buffer).

Maybe you can try to use just PBS and NP40 (0.1-1%) (without any phosphatase inhibitor NaF, Sodium orthovanadate, EDTA...)

-laurequillo-

Thanks, quiyo.

laurequillo on Dec 11 2009, 11:53 AM said:

EDTA is known to inhibit metalloproteases. May also chelate ions that are co-factors of certain enzymes (For example If you are gonna use CIP you should avoid EDTA in your buffer).

Maybe you can try to use just PBS and NP40 (0.1-1%) (without any phosphatase inhibitor NaF, Sodium orthovanadate, EDTA...)